Cytotoxic T cells in the cellular immune system are responsible for recognising cells that display “foreign” markings, and triggering an immunological response against such cells. Each cytotoxic T cell expresses a number of cell surface recognition receptors, which recognition receptors all possess precise specificity for a particular “foreign” peptide sequence, which recognition receptors are adapted to bind to HLA class I molecules expressed on the surface of cells scanned by the T cell. HLA class I molecules are cell surface molecules which possess a peptide binding groove exposed on the external surface of the cell, which groove is arranged under normal circumstances to bind a peptide derived from the interior of the cell. When a recognition receptor on a cytotoxic T cell binds to an HLA class I molecule on the surface of a scanned cell, the recognition receptor is enabled to contact the peptide binding groove of the HLA class I molecule and interact with any peptide contained therein. If this peptide matches the specificity of the recognition receptor, the T cell is said to recognise the scanned cell, and may consequently trigger an immunological response against said scanned cell.
Cytotoxic T cells of various specificities within a host immune system are also able to recognise and trigger an immunological response against a cell exhibiting an HLA class I molecule which is of a different allotype from the HLA class I molecules of the host cells. An immunological response of this kind is known as an “alloreactive” response.
An immunological response against a cell usually results in the lysis of the cell and/or the local release of cytokines. It has however been observed that cytotoxic T cells do not trigger the lysis of so-called antigen presenting cells (APCs) in this way. Instead, the immunological response triggered by T cell recognition of an HLA class I molecule on the surface of an antigen presenting cell results in the direct selective proliferation of the cytotoxic T cell. The host immune system consequently becomes immunised against any cells exhibiting the foreign peptide recognised by the surface recognition receptors on this T cell.
It is recognised that the effector mechanisms of the cellular immune system could be a powerful tool in the prevention and treatment of many illnesses, including malignant processes and infectious and auto-immune diseases, including cancer. A small number of the HLA class I molecules on a tumour cell surface may be found to bind peptides which are selectively expressed or over-expressed in tumour cells and are capable of being recognised by cytotoxic T cells in the immune system. Such peptides may furthermore be tumour specific, being found only infrequently, or not at all, on the HLA class I molecules of non-tumour cells. An example of one such tumour specific peptide is the HMW-MAA antigen found on melanoma cells. However, the number of HLA molecules presenting such peptides is generally too small to stimulate an effective immunological response against the tumour cell. Moreover, such peptides are rarely, if ever, presented by HLA class I molecules on the surface of APCs.
Attempts to enhance the response of the cellular immune system to tumour cells have hitherto focused on increasing tumour cell immunogenicity. In particular, various efforts have been made to produce high-level expression of immunogenic HLA class I molecules on the surface of tumour cells, through the techniques of gene therapy. The delivery of cDNA encoding an HLA class I gene containing an immunogenic peptide in the leader sequence of the HLA molecule has been described in Kang (Cancer Res. 57, 1997, 202-205). Meanwhile, Stopeck (J Clinical Oncolosv 15, 1997, 341-349) describes the transfection of allogeneic HLA class I in patients with melanoma. This work has demonstrated some response in clinical trials, but has also highlighted the difficulties involved in targeting tumour cells at multiple sites in vivo through the techniques of gene therapy.
Existing technology for production of alloreactive peptide specific cytotoxic T lymphocytes (CTLs) requires mixing donor cells with, initially, RMA cells (mouse cancer line), CIR or T2 cells (human tumour cell), and then Drosophila cells. This is both complicated and of low efficacy. These methods are also very ‘dirty’ which in this context means that they use cancer cells. It is undesirable to use cancer cells in the production of CTLs for administration to a subject.
Existing methods for producing CTLs in vitro are very labour intensive and expensive, involving culture of at least three different cell lines and complicated procedures of contacting the CTLs with each of these as part of the method(s). It is an advantageous feature of the present invention that CTL production is simplified and is also cheaper and/or easier and/or faster than existing techniques.
Subjects can be known to tolerate their own tumours rather than mounting an immunological response to them. This is because the tumour can appear as ‘own’ or ‘self’ to the immune system. Other people's immune systems (i.e. alloreactive) can fight better. However, the drawback with this approach is that a dual response can be produced in that the ordinary tissues of the recipient can be attacked too. This is the so-called graft versus host disease. The actual host can be attacked, which is problematic. One of the advantages of the present invention is the alleviation of this problem with existing methods by provision of better CTLs, which react with the target peptide in the appropriate context, such as when complexed with HLA-A2.
A number of approaches to the generation of anti-tumour CTLs, including use of dendritic cells, DNA vaccines and peptide administration are in clinical development, aiming to expand autologous tumour reactive CTLs. However producing autologous CTLs against certain targets is difficult or impossible due to immunological tolerance, and existing attempts at producing alloreactive CTLs have serious drawbacks as explained above (Sadovnikova 1998, Stauss 1999, Dutoit 2002).
Furthermore, some. target peptides apparently cannot be targeted by autologous means. Melan-A is an example of a peptide that can be targeted by autologous methods, but many others such as WT1 cannot be targeted by such methods. The present invention advantageously permits the targeting of a far wider range of peptides than conventional autologous methods.
Other techniques for production of CTLs have been described, such as production using T2 cells. T2 cells are a genetically altered human cell line, which is deficient in the genes encoding the transporter associated with antigen processing (TAP). This cell line therefore fails to properly load HLA-A2 Class I molecules with endogenous peptides. Therefore, exogenously added peptides can be made to bind a proportion of the HLA-A2 molecules on the surface of T2 cells. These techniques for producing CTLs rely on the culture of T2 cells, followed by peptide loading, cleaning of the cells and using them as a stimulatory cell to try to stimulate CTLs, for example from PBMC samples. Even when this labour intensive method appears to work, the stimulatory cells have to be constantly prepared and replaced at regular intervals in a rolling maintenance program. The present invention advantageously avoids the use of such T2 cells and also greatly simplifies the procedure. Furthermore, use of T2 cells is limited to HLA-A2, whereas the methods of the present invention are also applicable to other HLA types.
The existing technologies employed to produce alloreactive CTLs are relatively inefficient and the choice of target HLA class I is restricted by the limited allotypes of the commonly used antigen presenting cells, which are themselves genetically altered artificial cell lines.
It is desirable to separate out the beneficial effects of alloreactive transplants ‘graft versus leukaemia (tumour) effect’ from the unwanted ‘graft versus host disease’. Thus, the present invention relates to a method for the production of alloreactive peptide specific CTLs.
The production of alloreactive CTLs is advantageous over autologous CTLs because a subject may be tolerant to the autologous HLA peptide combination whilst tolerance in the alloreactive setting is much less likely. Thus the present invention relates to production of CTLs against a given HLA /peptide complex from a foreign donor (alloreactive) rather than autologous.
Without being bound by theory, it may be that some HLA types can produce better reactivity against certain other HLA types. For example, making an anti-HLA-A2 response may be easier for an HLA1 background but more difficult for an HLA4 background. The methods of the present invention may be advantageously applied to the study of this phenomenon.